This invention relates to chemically synthesized ribozymes, or enzymatic nucleic acid molecules and derivatives thereof.
The following is a brief description of ribozymes. This summary is not meant to be complete but is provided only for understanding of the invention that follows. This summary is not an admission that all of the work described below is prior art to the claimed invention.
Ribozymes are nucleic acid molecules having an enzymatic activity which is able to repeatedly cleave other separate RNA molecules in a nucleotide base sequence-specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage achieved in vitro (Zaug et al., 324, Nature 429 1986 ; Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic Acids Research 1371, 1989).
Because of their sequence-specificity, trans-cleaving ribozymes show promise as therapeutic agents for human disease (Usman and McSwiggen, 1995 Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med. Chem. 38, 2023-2037). Ribozymes can be designed to cleave specific RNA targets within the background of cellular RNA. Such a cleavage event renders the mRNA non-functional and abrogate protein expression from that RNA. In this manner, synthesis of a protein associated with a disease state can be selectively inhibited.
Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, since the effective concentration of ribozyme necessary to effect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability of the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base-pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base-pairing. Thus, it is thought that the specificity of action of a ribozyme is greater than that of antisense oligonucleotide binding the same RNA site.
Chemically-modified ribozymes can be synthesized which are stable in human serum for up to 260 hours (Beigelman et al., 1995 supra) and maintain near wild type (the chemically unmodified equivalent of a modified ribozyme) activity in vitro. A number of laboratories have reported that the enhanced cellular efficacy of phosphorothioate-substituted antisense molecules. The enhanced efficacy appears to result from either i) increased resistance to 5xe2x80x2-exonuclease digestion (De Clercq et al., 1970 Virology 42, 421-428; Shaw et al., 1991 Nucleic Acids Res. 19, 747-750), ii) intracellular localization to the nucleus (Marti et al., 1992 Antisense Res. Dev. 2, 27-39), or iii) sequence-dependent non-specific effects (Gao et al., 1992 Molec. Pharmac. 41, 223-229; Bock et al., 1992 Nature 355, 564-566; and Azad, et al., 1993 Antimicrob. Agents Chemother. 37, 1945-1954) which are not manifested in nonthioated molecules. Many effects of thioated compounds are probably due to their inherent tendency to associate non-specifically with cellular proteins such as the Sp1 transcription factor (Perez et al., 1994 Proc. Natl Acad Sci. U.S.A. 91, 5957-5961). Chemical modification of enzymatic nucleic acids that provide resistance to cellular 5xe2x80x2-exonuclease and 3xe2x80x2-exonuclease digestion without reducing the catalytic activity or cellular efficacy will be important for in vitro and in vivo applications of ribozymes.
Modification of oligonucleotides with a 5xe2x80x2-amino group offered resistance against 5xe2x80x2-exonuclease digestion in vitro (Letsinger and Mungall, 1970 J. Org. Chem. 35, 3800-3803).
Heidenreich et al., 1993 FASEB J. 7, 90 and Lyngstadaas et al., 1995 EMBO. J. 14, 5224, mention that hammerhead ribozymes with terminal phosphorothioate linkages can increase resistance against cellular exonucleases.
Seliger et al., Canadian Patent Application No. CA 2,106,819 and Prog. Biotechnol. 1994, 9 (EC B6: Proceedings Of The 6th European Congress On Biotechnology, 1993, Pt. 2), 681-4 describe xe2x80x9coligoribonucleotide and ribozyme analogs with terminal 3xe2x80x2-3xe2x80x2 and/or 5xe2x80x2-5xe2x80x2 internucleotide linkagesxe2x80x9d.
This invention relates to the incorporation of chemical modifications at the 5xe2x80x2 and/or 3xe2x80x2 ends of nucleic acids, which are particularly useful for enzymatic cleavage of RNA or single-stranded DNA. These terminal modifications are termed as either a 5xe2x80x2-cap or a 3xe2x80x2-cap depending on the terminus that is modified. Certain of these modifications protect the enzymatic nucleic acids from exonuclease degradation. Resistance to exonuclease degradation can increase the half-life of these nucleic acids inside a cell and improve the overall effectiveness of the enzymatic nucleic acids. These terminal modifications can also be used to facilitate efficient uptake of enzymatic nucleic acids by cells, transport and localization of enzymatic nucleic acids within a cell, and help achieve an overall improvement in the efficacy of ribozymes in vitro and in vivo.
The term xe2x80x9cchemical modificationxe2x80x9d as used herein refers to any base, sugar and/or phosphate modification that will protect the enzymatic nucleic acids from degradation by nucleases. Non-limiting examples of some of the chemical modifications and methods for their synthesis and incorporation in nucleic acids are described in FIGS. 7, 8, 11-16 and infra.
In a preferred embodiment, chemical modifications of enzymatic nucleic acids are featured that provide resistance to cellular 5xe2x80x2-exonuclease and/or 3xe2x80x2-exonuclease digestion without reducing the catalytic activity or cellular efficacy of these nucleic acids.
In a second aspect, the invention features enzymatic nucleic acids with 5xe2x80x2-end modifications (5xe2x80x2-cap) having the formula: 
wherein, X represents H, alkyl, amino alkyl, hydroxy alkyl, halo, trihalomethyl [CX3 (Xxe2x95x90Br, Cl, F)], N3, NH2, NHR, NR2 [each R is independently alkyl (C1-22), acyl (C1-22), or substituted (with alkyl, amino, alkoxy, halogen, or the like) or unsubstituted aryl], NO2, CONH2, COOR, SH, OR, ONHR, PO42xe2x88x92, PO3S2xe2x88x92, PO2S22xe2x88x92, POS32xe2x88x92, PO3NH2xe2x88x92, PO3NHRxe2x88x92, NO2, CONH2, COOR, B represents a natural base or a modified base or H; Y represents rest of the enzymatic nucleic acid; and R1 represents H, O-alkyl, C-alkyl, halo, NHR, or OCH2SCH3 (methylthiomethyl). The 5xe2x80x2-modified sugar synthesis is as described by Moffatt, in Nucleoside Analogues:Chemistry, Biology and Medical Applications, Walker, DeClercq, and Eckstein, Eds,; Plenum Press:New York, 1979, pp 71 (incorporated by reference herein).
Another preferred embodiment of the invention features enzymatic nucleic acid molecules having a 5xe2x80x2-cap, wherein said cap is selected from but not limited to, a group comprising, 4xe2x80x2,5xe2x80x2-methylene nucleotide; 1-(xcex2-D-erythrofuranosyl) nucleotide; 4xe2x80x2-thio nucleotide, carbocyclic nucleotide; 5xe2x80x2-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; xcex1-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3xe2x80x2,4xe2x80x2-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5xe2x80x2-5xe2x80x2-inverted nucleotide moeity; 5xe2x80x2-5xe2x80x2-inverted abasic moeity; 5xe2x80x2-phosphoramidate; 5xe2x80x2-phosphorothioate; 1,4-butanediol phosphate; 5xe2x80x2-amino; bridging and/or non-bridging 5xe2x80x2-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridging or non bridging methylphosphonate and 5xe2x80x2-mercapto moeities (for more details see Beaucage and lyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).
In a third aspect, the invention features enzymatic nucleic acids with 3xe2x80x2-end modifications (3xe2x80x2-cap) having the formula: 
wherein, X represents 4xe2x80x2-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; xcex1-nucleotides; modified base nucleotide; phosphorodithioate linkage; threopentofuranosyl nucleotide; acyclic 3xe2x80x2,4xe2x80x2-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3xe2x80x2-3xe2x80x2-inverted nucleotide moeity; 3xe2x80x2-3xe2x80x2-inverted abasic moeity; 3xe2x80x2-2xe2x80x2-inverted nucleotide moeity; 3xe2x80x2-2xe2x80x2-inverted abasic moeity; 1,4-butanediol; 3xe2x80x2-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3xe2x80x2-phosphate; 3xe2x80x2-phosphorothioate; or bridging or nonbridging methylphosphonate moeity; B represents a natural base or a modified base or H; Y represents rest of the enzymatic nucleic acid; and R1 represents H, O-alkyl, C-alkyl, halo, NHR [R=alkyl (C1-22), acyl (C1-22), substituted or unsubstituted aryl], or OCH2SCH3 (methylthiomethyl).
In yet another preferred embodiment the invention features enzymatic nucleic acid molecules having a 3xe2x80x2-cap, wherein said cap is selected from but not limited to, a group comprising, 4xe2x80x2-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; a-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3xe2x80x2,4xe2x80x2-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3xe2x80x2-3xe2x80x2-inverted nucleotide moeity; 3xe2x80x2-3xe2x80x2-inverted abasic moeity; 3xe2x80x2-2xe2x80x2-inverted nucleotide moeity; 3xe2x80x2-2xe2x80x2-inverted abasic moeity; 1,4-butanediol phosphate; 3xe2x80x2-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3xe2x80x2-phosphate; 3xe2x80x2-phosphorothioate; phosphorodithioate; or bridging or nonbridging methylphosphonate moeity (for more details see Beaucage and lyer, 1993, Tetrahedron 49, 1925; incorporated by reference herein).
In a fourth aspect, the invention features enzymatic nucleic acids with both 5xe2x80x2-cap and a 3xe2x80x2-cap which may be same or different.
The term xe2x80x9cnucleotidexe2x80x9d is used as recognized in the art to include natural bases, and modified bases well known in the art. Such bases are generally located at the 1xe2x80x2 position of a sugar moiety. Nucleotide generally comprise a base, sugar and a phosphate group. The nucleotide can be unmodified or modified at the sugar, phosphate and/or base moeity. The term xe2x80x9cabasicxe2x80x9d or xe2x80x9cabasic nucleotidexe2x80x9d as used herein encompasses sugar moieties lacking a base or having other chemical groups in place of base at the 1xe2x80x2 position.
By the phrase xe2x80x9cenzymatic nucleic acidxe2x80x9d is meant a catalytic modified-nucleotide-containing nucleic acid molecule that has complementarity in a substrate binding region to a specified gene target, and also has an enzymatic activity that specifically cleaves RNA or DNA in that target. That is, the enzymatic nucleic acid is able to intramolecularly or intermolecularly cleave RNA or DNA and thereby inactivate a target RNA or DNA molecule. This complementarity functions to allow sufficient hybridization of the enzymatic RNA molecule to the target RNA or DNA to allow the cleavage to occur. 100% Complementarity is preferred, but complementarity as low as 50-75% may also be useful in this invention. The nucleic acids may be modified at the base, sugar, and/or phosphate groups. The term enzymatic nucleic acid is used interchangeably with phrases such as ribozymes, catalytic RNA, enzymatic RNA, catalytic DNA, nucleozyme, DNAzyme, RNA enzyme, endoribonuclease, minizyme, leadzyme or DNA enzyme. All of these terminologies describe nucleic acid molecules with enzymatic activity.
There are several examples of modified bases as it relates to nucleic acids, is well known in the art and has recently been summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the non-limiting examples of base modifications that can be introduced into enzymatic nucleic acids without significantly effecting their catalytic activity include, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine); Guanosine or adenosine residues may be replaced by diaminopurine residues in either the core or stems.
There are several examples in the art describing sugar modifications that can be introduced into enzymatic nucleic acid molecules without significantly effecting catalysis and significantly enhancing their nuclease stability and efficacy. Sugar modification of enzymatic nucleic acid molecules have been extensively described in the art (see Eckstein et al., International Publication PCT No. WO 92/07065; Perrault et al. Nature 1990, 344,565-568; Pieken et al. Science 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem. Sci. 1992, 17, 334-339; Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995 J. Biol. Chem. 270, 25702). Such publications describe the location of incorporation of modifications and the like, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein.
Specifically, an xe2x80x9calkylxe2x80x9d group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably it is a lower alkyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, xe2x95x90O, xe2x95x90S, NO2 or N(CH3)2, amino, or SH. The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12 carbons. More preferably it is a lower alkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, xe2x95x90O, xe2x95x90S, NO2, halogen, N(CH3)2, amino, or SH. The term xe2x80x9calkylxe2x80x9d also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has 1 to 12 carbons. More preferably it is a lower alkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted the substituted group(s) is preferably, hydroxyl, cyano, alkoxy, xe2x95x90O, xe2x95x90S, NO2 or N(CH3)2, amino or SH.
Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An xe2x80x9carylxe2x80x9d group refers to an aromatic group which has at least one ring having a conjugated xcfx80 electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted. The preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An xe2x80x9calkylarylxe2x80x9d group refers to an alkyl group (as described above) covalently joined to an aryl group (as described above. Carbocyclic aryl groups are groups wherein the ring atoms on the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An xe2x80x9camidexe2x80x9d refers to an xe2x80x94C(O)xe2x80x94NHxe2x80x94R, where R is either alkyl, aryl, alkylaryl or hydrogen. An xe2x80x9cesterxe2x80x9d refers to an xe2x80x94C(O)xe2x80x94ORxe2x80x2, where R is either alkyl, aryl, alkylaryl or hydrogen.
The 5xe2x80x2-cap and/or 3xe2x80x2-cap derivatives of this invention provide enhanced activity and stability to the enzymatic nucleic acids containing them.
By xe2x80x9ccomplementarityxe2x80x9d is meant a nucleic acid that can form hydrogen bond(s) with other RNA sequence by either traditional Watson-Crick or other non-traditional types (for example, Hoogsteen type) of base-paired interactions.
By xe2x80x9cbridgingxe2x80x9d and xe2x80x9cnonbridgingxe2x80x9d are meant to indicate the relative positions of oxygen atom involved in the formation of standard phosphodiester linkage in a nucleic acid. These backbone oxygen atoms can be readily modified to impart resistance against nuclease digestion. The terms are further defined as follows: 
wherein xe2x80x9cxxe2x80x9d is bridging oxygen and xe2x80x98yxe2x80x99 is nonbridging oxygen.
In preferred embodiments of this invention, the enzymatic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis delta virus (HDV), group I intron, RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs are described by Rossi et a., 1992, Aids Research and Human Retroviruses 8, 183, of hairpin motifs by Hampel et a/., EP0360257, Hampel and Tritz, 1989 Biochemistry 28,4929, and Hampel et al., 1990 Nucleic Acids Res. 18, 299, and an example of the hepatitis delta virus motif is described by Perrotta and Been, 1992 Biochemistry 31, 16; of the RNaseP motif by Guerrier-Takada et al., 1983 Cell 35, 849 and Forster and Altman, 1990 Science 249, 783, Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, 1990 Cell 61, 685-696; Saville and Collins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Guo and Collins, 1995 EMBO J. 14, 368) and of the Group I intron by Cech et a/., U.S. Pat. No. 4,987,071. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
The invention provides a method for producing a class of enzymatic cleaving agents which exhibit a high degree of specificity for the RNA of a desired target. The enzymatic nucleic acid molecule is preferably targeted to a highly conserved sequence region of a target such that specific treatment of a disease or condition can be provided with a single enzymatic nucleic acid. Such enzymatic nucleic acid molecules can be delivered exogenously to specific cells as required. In the preferred hammerhead motif the small size (less than 60 nucleotides, preferably between 30-40 nucleotides in length) of the molecule allows the cost of treatment to be reduced compared to other ribozyme motifs.
Synthesis of nucleic acids greater than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small enzymatic nucleic acid motifs (e.g., of the hammerhead structure) are used for exogenous delivery. The simple structure of these molecules increases the ability of the enzymatic nucleic acid to invade targeted regions of the mRNA structure. Unlike the situation when the hammerhead structure is included within longer transcripts, there are no non-enzymatic nucleic acid flanking sequences to interfere with correct folding of the enzymatic nucleic acid structure or with complementary regions.
Therapeutic ribozymes must remain stable within cells until translation of the target mRNA has been inhibited long enough to reduce the levels of the undesirable protein. This period of time varies between hours to days depending upon the disease state. Clearly, ribozymes must be resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of RNA (Wincott et al., 1995 Nucleic Acids Res. 23, 2677; incorporated by reference herein) have expanded the ability to modify ribozymes to enhance their nuclease stability. The majority of this work has been performed using hammerhead ribozymes (reviewed in Usman and McSwiggen, 1995 supra) and can be readily extended to other ribozyme motifs.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.